建立一种同时鉴别诊断猪瘟病毒（classical swine fever virus, CSFV)、非洲猪瘟病毒（African swine fever virus, ASF）和猪非典型瘟病毒（atypical porcine pestivirus, APPV）的快速、灵敏、有效的检测方法。依据GenBank中登录的CSFV (5¢ UTR)、ASFV (B646L) 和 APPV (5¢ UTR) 的高度保守基因序列分别设计和优化了多对特异性引物和Taq-man探针，以保守区基因序列分别制备三种阳性质粒，用矩阵法优化单重/多重荧光PCR的反应体系和条件，为避免荧光通道的交叉干扰多重荧光PCR扩增，结合所标记的荧光报告基团做颜色补偿试验，构建标准曲线的扩增图和对应的线性方程，并进行特异性、敏感性、重复性、符合性以及临床样本的检测等试验。三种病毒的标准曲线相关系数均达到0.995以上，具有良好的线性关系；与其它常见猪病无交叉扩增反应，具有很好的特异性；多重荧光PCR的最低检测量均为1 copy/mL，具有较高的敏感性；组内和组间的变异系数均小于1%，具有很好的重复性。该方法与CSF的国标(GB/T 27540-2011), ASF的国标 (GB/T 18648-2020)，APPV的发明专利 (CN108611442A)检测样本盘的22个毒株样本符合率为100%。本研究建立的多重荧光PCR检测方法具有快速、高效、通量高、特异性好、灵敏度高等特点，可以对CSFV、ASFV和APPV病毒进行鉴别检测，为动物疫病的流行病学调查、疫情的检测提供一种新型的检测手段。本研究结合荧光PCR仪不同荧光通道设计CSFV、ASFV和APPV探针荧光信号强度较高且干扰较小的FAM、CY5和HEX报告基团，建立多重荧光PCR检测方法，用于同时鉴别诊断3种主要猪病毒的检测方法，在临床诊断中具有重要的应用价值。

Classical swine fever caused by Classical swine fever virus (CSFV) is a serious problem for swine industries in developing countries, which successful control of the disease have been relying on vaccination. However, classical swine fever still occurs in some immunized swine herds for various reasons. In this study, we conducted animal experiments to examine the influence of single or mixed infection with Porcine parvo virus (PPV), Pseudorabies virus (PRV) and Porcine reproductive and respiratory syndrome virus (PRRSV) on the protective immunity induced by the Lapinized hog cholera virus (HCLV) vaccine and the pathogenicity of CSFV. In experiment 1, pigs were first inoculated with PPV, PRV or PRRSV, then immunized with HCLV, and finally challenged with a highly virulent CSFV Shimen strain. All of the pigs immunized with HCLV survived after the challenge, while all of the pigs in the non-immunized control group died after the challenge. The pigs in the group immunized with HCLV did not show any clinical symptoms of classical swine fever and were negative with CSFV after the challenge. The pigs infected with the non-CSFV before HCLV immunization did not display any clinical symptoms after the challenge with CSFV Shiman strain, but 11 of the 12 pigs were positive with CSFV. In experiment 2, pre-infections with PPV, PRV, and PRRSV were followed by inoculation with a low-virulence CSFV strain (CSFV 39), and then the pigs were challenged with the CSFV Shimen strain. Infections by either PPV, PRV or PRRSV did not enhance the virulence of CSFV-39, but pigs infected by a mixture of the 3 viruses developed clinical symptoms after inoculation with CSFV-39. The mixed infection also increased mortality caused by the challenge with the CSFV Shimen strain. Together, these results showed PPV, PRV and PRRSV infections in pigs can reduce the efficacy of the HCLV vaccine and enhance the pathogenicity of CSFV, which may partly explain the immunization failure against CSFV in some swine herds.